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Claims  |
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What is claimed is:
1. A device for amplifying a preselected polynucleotide in a sample by
conducting a polynucleotide amplification reaction, the device comprising:
a solid substrate which is microfabricated to define:
a sample inlet port;
a mesoscale flow system comprising:
a sample flow channel extending from said inlet port; and
a polynucleotide amplification reaction chamber, in fluid communication
with said flow channel;
said sample flow channel and said reaction chamber having at least one
cross-sectional dimension of width or depth which is about 0.1 to 500
.mu.m, said chamber and said channel being of dissimilar dimension; and
a fluid exit port in fluid communication with said flow system; and
means for thermally cycling the contents of said chamber whereby in each
cycle the temperature is controlled to dehybridize double stranded
polynucleotide and to permit the in vitro amplification of a preselected
polynucleotide.
2. The device of claim 1, wherein said amplification reaction is a
polymerase chain reaction (PCR), and wherein said amplification chamber
contains: a preselected polynucleotide, a polymerase, nucleoside
triphosphates, a first primer hybridizable with said sample
polynucleotide, and a second primer hybridizable with a nucleic acid
comprising a sequence complementary to said polynucleotide, wherein said
first and second primers define the termini of the polynucleotide product
of the polymerization reaction; and
wherein said means for thermally cycling comprises means for thermally
cycling the contents of said chamber between a temperature controlled to
dehybridize double stranded polynucleotide thereby to produce single
stranded polynucleotide, to permit annealing of said primers to
complementary regions of single stranded polynucleotide, and to permit
synthesis of polynucleotide between said primers, thereby to amplify said
preselected polynucleotide.
3. The device of claim 1 wherein said amplification chamber comprises:
a first section at a temperature which dehybridizes double stranded
polynucleotide;
a second section at a temperature which permits annealing of complementary
regions of single stranded polynucleotide and permits amplification of
polynucleotide;
a flow path disposed between said first and second sections; and
wherein said device includes:
means for repeatedly transporting the contents of said chamber between at
least said first and said second sections to implement plural cycles of
amplification of said polynucleotide.
4. The device of claim 3
wherein said second section and said flow path are spaced apart from said
first section such that upon transport of the contents of said chamber
from said first section to said second section, the sample cools passively
to a temperature sufficient to permit annealing of single stranded
polynucleotide and to permit amplification of said preselected
polynucleotide.
5. The device of claim 3 further comprising means for separately thermally
controlling said first and said second sections.
6. The device of claim 4 further comprising means for thermally controlling
said first section.
7. The device of claim 5 or 6 wherein said means for thermally controlling
comprises electrical resistance means in said substrate.
8. The device of claim 5 or 6 wherein said means for thermally controlling
comprises means for providing electromagnetic energy to said amplification
chamber.
9. The device of claim 1 wherein said solid substrate comprises
microfabricated silicon.
10. The device of claim 1 further comprising an appliance for use in
combination with said substrate, said appliance comprising:
means for holding said substrate; and
fluid input means interfitting with an inlet port on said substrate.
11. The device of claim 10 further comprising pump means for passing fluid
through the flow system of said substrate when it is held in said holding
means.
12. The device of claim 11 wherein said appliance further comprises a
reagent reservoir and means for delivering a reagent to said flow system.
13. The device of claim 10 wherein said means for thermally cycling
comprises an electromagnetic energy source; and
wherein said electromagnetic energy source is provided in said appliance.
14. A device for amplifying a preselected polynucleotide in a sample, the
device comprising:
a solid substrate microfabricated to define:
a sample inlet port;
a mesoscale flow system comprising:
a sample flow channel extending from said inlet port; and
a polynucleotide amplification reaction chamber, in fluid communication
with said flow channel, containing a preselected polynucleotide and
polynucleotide amplification reagents;
said sample flow channel and said reaction chamber having at least one
cross-sectional dimension of width or depth which is about 0.1 to 500
.mu.m, said chamber and said channel being of dissimilar dimension; and
a fluid exit port in fluid communication with said flow system; and
means for thermally cycling the contents of said chamber whereby, in each
cycle, temperature is controlled to dehybridize double stranded
polynucleotide, and to permit synthesis of polynucleotide, thereby to
amplify said preselected polynucleotide.
15. The device of claim 14 wherein said flow system further comprises a
detection chamber in fluid communication with said amplification chamber.
16. The device of claim 14 wherein said amplification chamber comprises:
a first section at a temperature which dehybridizes double stranded
polynucleotide;
a second section at a temperature which permits annealing of single
stranded polynucleotide, and which permits amplification of
polynucleotide; and
a flow path disposed between said first and second sections; and
wherein the device further comprises means for repeatedly transporting the
contents of said chamber between said first and said second sections to
implement plural cycles of amplification of said polynucleotide.
17. The device of claim 14 further comprising an appliance for use in
combination with said substrate, said appliance comprising:
a nesting site for holding said substrate, which comprises fluid input
means interfitting with an inlet port on said substrate.
18. The device of claim 17 wherein said device includes electrical contacts
fabricated in the substrate; and
wherein said nesting site further includes an electrical connection for
interfitting with said electrical contact in said substrate.
19. The device of claim 17 wherein the appliance further comprises pump
means for passing fluid through the flow system of said substrate when it
is held in said holding means.
20. The device of claim 17 wherein the appliance further comprises a power
supply.
21. A method for amplifying a preselected polynucleotide in a sample by
conducting a polynucleotide amplification reaction, the method comprising:
(i) providing a device comprising:
a solid substrate microfabricated to define:
a sample inlet port;
a mesoscale flow system comprising:
a sample flow channel extending from said inlet port; and
a polynucleotide amplification reaction chamber in fluid communication with
said flow channel;
said sample flow channel and said reaction chamber having at least one
cross-sectional dimension of width or depth which is about 0.1 to 500
.mu.m, said chamber and said channel being of dissimilar dimension; and
a fluid exit port in fluid communication with said flow system; and
means for thermally regulating the contents of said chamber at a
temperature controlled to permit amplification of said preselected
polynucleotide;
(ii) delivering, through said inlet port and said mesoscale flow system to
said reaction chamber, a sample polynucleotide and reagents required for
an in vitro polynucleotide amplification reaction; and
(iii) thermally controlling the contents of said chamber to permit
amplification of said polynucleotide.
22. The method of claim 31 wherein said amplification reaction is a
polymerase chain reaction (PCR);
wherein, in step (i), said means for thermally controlling comprises means
for thermally cycling the contents of said chamber;
wherein step (ii) includes the step of adding to said amplification
chamber: a polymerase, nucleoside triphosphates, a first primer
hybridizable with said sample polynucleotide, and a second primer
hybridizable with a nucleic acid comprising a sequence complementary to
said polynucleotide, and wherein said first and second primers define the
termini of the polynucleotide product of the polymerization reaction; and
wherein step (iii) includes the step of thermally cycling the contents of
said chamber whereby, in each cycle, the temperature is controlled to
dehybridize double stranded polynucleotide thereby to produce single
stranded polynucleotide, to permit annealing of complementary regions of
single stranded polynucleotide, and to permit synthesis and polymerization
of polynucleotide between said primers.
23. The method of claim 22 wherein said amplification chamber comprises:
a first section at a temperature which dehybridizes double stranded
polynucleotide;
a second section at a temperature which permits annealing of complementary
regions of single stranded polynucleotide and permits amplification of
polynucleotide;
a flow path disposed between said first and second sections;
wherein the device further includes:
means for repeatedly transporting the contents of said chamber between said
first and said second sections; and
wherein step (iii) includes the step of repeatedly transporting the
contents of said chamber between said first and said second sections to
implement plural cycles of amplification of polynucleotide.
24. The method of claim 23 wherein said first section is controlled at a
temperature to dehybridize double stranded polynucleotide; and
wherein said second section and said flow path are spaced apart from said
first section such that upon transport of the contents of said chamber
from said first section to said second section, the sample cools
substantially to a temperature to anneal and polymerize; and
wherein step (iii) includes the step of repeatedly transporting the
contents of said chamber between said first and second sections to
polymerize said polynucleotide.
25. A device for amplifying a preselected polynucleotide in a sample by
conducting a polynucleotide amplification reaction, the device comprising:
a solid substrate microfabricated to define:
a sample inlet port;
a mesoscale flow system comprising:
a sample flow channel extending from said inlet port; and
a polynucleotide amplification chamber, in fluid communication with said
flow channel, said chamber containing reagents for amplifying a
preselected polynucleotide in vitro;
said sample flow channel and said reaction chamber having at least one
cross-sectional dimension of width or depth which is about 0.1 to 500
.mu.m, said chamber and said channel being of dissimilar dimension; and
a fluid exit port in fluid communication with said flow system.
26. The device of claim 25 wherein said reagents comprise reagents for
conducting a polymerase chain reaction.
27. The device of claim 25 wherein said reagents comprise reagents for
conducting a ligase chain reaction. |
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Claims |
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Description |
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REFERENCE TO RELATED APPLICATIONS
Ths application is being filed contemporaneously with the following related
applications: U.S. Ser. No. 07/877,702, filed May 1, 1992, abandoned; U.S.
Ser. No. 07/877,701, filed May 1, 1992, abandoned; U.S. Ser. No.
07/877,536 filed May 1, 1992, now U.S. Pat. No. 5,304,487; and U.S. Ser.
No. 07/877,661, filed May 1, 1992, now U.S. Pat. No. 5,296,375; the
disclosures of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
This invention relates generally to methods and apparatus for conducting
analyses. More particularly, the invention relates to the design and
construction of small, typically single-use, modules capable of analyses
involving polymerase chain reaction (PCR).
In recent decades the art has developed a very large number of protocols,
test kits, and cartridges for conducting analyses on biological samples
for various diagnostic and monitoring purposes. Immunoassays,
agglutination assays, and analyses based on polymerase chain reaction,
various ligand-receptor interactions, and differential migration of
species in a complex sample all have been used to determine the presence
or concentration of various biological compounds or contaminants, or the
presence of particular cell types.
Recently, small, disposable devices have been developed for handling
biological samples and for conducting certain clinical tests. Shoji et al.
reported the use of a miniature blood gas analyzer fabricated on a silicon
wafer. Shoji et al., Sensors and Actuators, 15:101-107 (1988). Sato et al.
reported a cell fusion technique using micromechanical silicon devices.
Sato et al., Sensors and Actuators, A21-A23:948-953 (1990). Ciba Corning
Diagnostics Corp. (USA) has manufactured a microprocessor-controlled laser
photometer for detecting blood clotting.
Micromachining technology originated in the microelectronics industry.
Angell et al., Scientific American, 248:44-55 (1983). Micromachining
technology has enabled the manufacture of microengineered devices having
structural elements with minimal dimensions ranging from tens of microns
(the dimensions of biological cells) to nanometers (the dimensions of some
biological macromolecules). This scale is referred to herein as
"mesoscale". Most experiments involving mesoscale structures have involved
studies of micromechanics, i.e., mechanical motion and flow properties.
The potential capability of mesoscale structures has not been exploited
fully in the life sciences.
Brunette (Exper. Cell Res., 167:203-217 (1986) and 164:11-26 (1986))
studied the behavior of fibroblasts and epithelial cells in grooves in
silicon, titanium-coated polymers and the like. McCartney et al. (Cancer
Res., 41:3046-3051 (1981)) examined the behavior of tumor cells in grooved
plastic substrates. LaCelle (Blood Cells, 12:179-189 (1986)) studied
leukocyte and erythrocyte flow in microcapillaries to gain insight into
microcirculation. Hung and Weissman reported a study of fluid dynamics in
micromachined channels, but did not produce data associated with an
analytic device. Hung et al., Med. and Biol. Engineering, 9:237-245
(1971); and Weissman et al., Am. Inst. Chem. Eng. J., 17:25-30 (1971).
Columbus et al. utilized a sandwich composed of two orthogonally
orientated v-grooved embossed sheets in the control of capillary flow of
biological fluids to discrete ion-selective electrodes in an experimental
multi-channel test device. Columbus et al., Clin. Chem., 33:1531-1537
(1987). Masuda et al. and Washizu et al. have reported the use of a fluid
flow chamber for the manipulation of cells (e.g. cell fusion). Masuda et
al., Proceedings IEEE/IAS Meeting, pp. 1549-1553 (1987); and Washizu et
al., Proceedings IEEE/IAS Meeting pp. 1735-1740 (1988). The art has not
fully explored the potential of using mesoscale devices for the analyses
of biological fluids.
Methodologies for using polymerase chain reaction (PCR) to amplify a
segment of DNA are well established. (See e.g., Maniatis et al. Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989,
pp. 14.1-14.35.) A PCR amplification reaction can be performed on a DNA
template using a thermostable DNA polymerase, e.g., Taq DNA polymerase
(Chien et al. J. Bacteriol.:127:1550 (1976)), nucleoside triphosphates,
and two oligonucleotides with different sequences, complementary to
sequences that lie on opposite strands of the template DNA and which flank
the segment of DNA that is to be amplified ("primers"). The reaction
components are cycled between a higher temperature (e.g., 94.degree. C.)
for dehybridizing ("melting") double stranded template DNA, followed by a
lower temperature (e.g., 65.degree. C.) for annealing and polymerization.
A continual reaction cycle between dehybridization, annealing and
polymerization temperatures provides exponential amplification of the
template DNA. For example, up to 1 .mu.g of target DNA up to 2 kb in
length can be obtained from 30-35 cycles of amplification with only
10.sup.-6 .mu.g of starting DNA. Machines for performing automated PCR
chain reactions using a thermal cycler are available (Perkin Elmer Corp.)
PCR amplification has been applied to the diagnosis of genetic disorders
(Engelke et al., Proc. Natl. Acad. Sci., 85:544 (1988), the detection of
nucleic acid sequences of pathogenic organisms in clinical samples (Ou et
al., Science, 239:295 (1988)), the genetic identification of forensic
samples, e.g., sperm (Li et al., Nature, 335:414 (1988)), the analysis of
mutations in activated oncogenes (Farr et al., Proc. Natl. Acad. Sci.,
85:1629 (1988)) and in many aspects of molecular cloning (Oste,
BioTechniques, 6:162 (1988)). PCR assays can be used in a wide range of
applications such as the generation of specific sequences of cloned
double-stranded DNA for use as probes, the generation of probes specific
for uncloned genes by selective amplification of particular segments of
cDNA, the generation of libraries of cDNA from small amounts of mRNA, the
generation of large amounts of DNA for sequencing, and the analysis of
mutations. There is a need for convenient rapid systems for PCR analyses,
which could be used clinically in a wide range of potential applications
in clinical tests such as tests for paternity, and genetic and infectious
diseases.
An object of the invention is to provide analytical systems with optimal
reaction environments that can analyze microvolumes of sample, detect very
low concentrations of a polynucleotide, and produce analytical results
rapidly. Another object is to provide easily mass produced, disposable,
small (e.g., less than 1 cc in volume) devices having mesoscale functional
elements capable of rapid, automated PCR analyses of a preselected cell or
cell-free sample, in a range of applications. It is a further object of
the invention to provide a family of such devices that individually can be
used to implement a range of rapid clinical tests, e.g., tests for viral
or bacterial infection, tests for cell culture contaminants, or tests for
the presence of recombinant DNA or a gene in a cell, and the like.
SUMMARY OF THE INVENTION
The invention provides a family of small, mass produced, typically one-use
devices for conducting a polynucleotide polymerization reaction to enable
the rapid amplification of a polynucleotide in a sample. In one
embodiment, the device comprises a solid substrate, on the order of a few
millimeters thick and approximately 0.2 to 2.0 centimeters square, that is
microfabricated to define a sample inlet port and a mesoscale flow system.
The flow system of the device includes a sample flow channel extending
from the inlet port, and a polynucleotide polymerization reaction chamber
in fluid communication with the flow channel polynucleotide. The term
"mesoscale" is used herein to define chambers and flow passages having a
cross-sectional dimension on the order of 0.1 .mu.m to 500 .mu.m, with
preferred reaction chamber widths on the order of 2.0 to 500 .mu.m, more
preferably 3-100 .mu.m. For many applications, channels of 5-50 .mu.m
widths will be useful. Chambers in the substrate wherein amplification
takes place may have somewhat larger dimensions, e.g., 1-5 mm. Preferred
reaction chamber and channel depths are on the order of 0.1 to 100 .mu.m,
typically 2-50 .mu.m. The flow channels in the devices, leading to the
reaction chambers, have preferred widths on the order of 2.0 to 200 .mu.m
and depths on the order of 0.1 to 100 .mu.m.
In one embodiment, the devices may be utilized to implement a
polymerization chain reaction (PCR) in the reaction chamber. The reaction
chamber may be provided with reagents for PCR including a sample
polynucleotide, polymerase, nucleoside triphosphates, a first primer
hybridizable with the sample polynucleotide, and a second primer
hybridizable with a sequence that is complementary to the sample
polynucleotide, wherein the first and second primers define the termini of
the polymerized polynucleotide product. The device also may include means
for thermally cycling the contents of the PCR chamber, such that, in each
cycle, the temperature is controlled to 1) dehybridize ("melt") double
stranded polynucleotide, 2) anneal the primers to single stranded
polynucleotide, and 3) synthesize amplified polynucleotide between the
primers. In one embodiment, the PCR chamber may comprise one section which
is thermally cycled sequentially to the required temperatures for PCR.
Alternatively, the PCR chamber may comprise two or more sections, set at
the different temperatures required for dehybridization, annealing and
polymerization, in which case the device further comprises means for
cycling the contents of the chamber between the sections to implement the
PCR, e.g., a pump or other means as disclosed herein. The device may
further include means for detecting the amplified polynucleotide. The
devices may be used to implement a variety of automated, sensitive and
rapid polynucletide analyses, including analyses for the presence of
polynucleotides in cells or in solution, or for analyses for a virus or
cell types using the presence of a particular polynucleotide as a marker.
Generally, as disclosed herein, the solid substrate comprises a chip,
containing the mesoscale flow system and the reaction chamber(s). The
mesoscale flow channels and reaction chambers may be designed and
fabricated from silicon and other solid substrates using established
micromachining methods. The mesoscale flow systems in the devices may be
constructed by microfabricating flow channels and one or more reaction
chambers into the surface of the substrate, and then adhering a cover,
e.g., a transparent glass cover, over the surface. The devices analyze
microvolumes (<10 .mu.L) of a sample, introduced into the flow system
through an inlet port defined, e.g., by a hole communicating through the
substrate or the cover. The volume of the mesoscale flow system typically
will be <5 .mu.L, and the volume of individual channels, chambers, or
other functional elements are often less than 1 .mu.L, e.g., in the
nanoliter or even picoliter range. Polynucleotides present in very low
concentrations, (e.g. nanogram quantities) can be rapidly amplified (<10
minutes) and detected. After a polynucleotide polymerization assay is
complete, the devices may be discarded.
The chips typically will be used with an appliance which contains a nesting
site for holding the chip, and which mates one or more input ports on the
chip with one or more flow lines in the appliance. After a biological
fluid sample suspected to contain a particular polynucleotide is applied
to the inlet port of the substrate, the chip is placed in the appliance
and a pump, e.g., in the appliance, is actuated to force the sample
through the flow system. Alternatively, a sample may be injected into the
chip by the appliance. Reagents required for the assay, such as a
polymerase, may be added to the polynucleotide sample prior to injection
into the chip. Alternatively, reagents necessary to complete the assay can
be injected into the reaction chamber from a separate inlet port, e.g., by
the appliance. Fluid samples and reagents may also enter the mesoscale
flow system by capillary action.
In one embodiment, the devices may be utilized to perform a PCR assay, and
the temperature of one or more section(s) in the reaction chamber can be
regulated by, e.g., providing one or more electrical resistance heaters in
the substrate near the reaction chamber, or by using a pulsed laser or
other source of electromagnetic energy directed to the reaction chamber.
The appliance may include electrical contacts in the nesting region which
mate with contacts integrated into the structure of the chip, e.g., to
power electrical resistance heating of the reaction chamber. A cooling
element may also be provided in the appliance to assist in the thermal
regulation of the reaction chamber. The appliance may be provided with
conventional circuitry sensors in communication with sensors in the device
for thermally regulating the PCR temperature cycles required for the
dehybridization and polymerization reactions.
The amplified polynucleotide produced by the polynucleotide amplification
reaction in the mesoscale reaction chamber can be collected through a port
in the substrate and detected, e.g., by gel electrophoresis or any other
method. Alternatively, a mesoscale detection region may be microfabricated
in the substrate, in fluid communication with the reaction chamber in the
device, as a part of the mesoscale flow system. The detection region may
include a labeled binding moiety, such as a labeled polynucleotide or
antibody probe, capable of detectably binding with the amplified
polynucleotide. The presence of polymerized polynucleotide product in the
detection region can be detected, e.g., by optical detection of
agglutination of the polymerized polynucleotide and the binding moiety
through a glass cover over the detection region or through a translucent
section of the substrate itself.
A positive assay may also be indicated by detectable changes in sample
fluid flow properties such as changes in pressure or electrical
conductivity at different points in the flow system upon production of
polymerized polynucleotide in the reaction chamber. In one embodiment, the
device comprises a mesoscale flow system which includes a polynucleotide
amplification reaction chamber, and a detection region is used in
combination with an appliance which includes sensing equipment such as a
spectrophotometer capable of reading a positive result through an optical
window, e.g., disposed over the detection region. The appliance may also
be designed to receive electrical signals indicative of a pressure
reading, conductivity, or the like, sensed in the reaction chamber, the
detection region, or some other region of the flow system.
The substrate may comprise a plurality of detection/reaction chambers to
enable the rapid parallel detection of polynucleotides in a mixture. The
mesoscale flow system may include protrusions, or a section of reduced
cross sectional area, to enable the lysis of cells in the microsample
prior to delivery to the reaction chamber. Sharp edged pieces of silicon,
trapped in the flow path, can also be used as a lysis means. The mesoscale
flow system also may include a cell capture region comprising a binding
moiety, e.g., immobilized on a wall of a flow channel, which binds a
particular type of cell in a heterogeneous cell population at a low fluid
flow rate, and at a greater flow rate, releases the cell type prior to
delivery of the cells to a cell lysis region then to a reaction chamber.
In this embodiment, intracellular DNA or RNA is isolated from a selected
cell subpopulation and delivered to the mesoscale reaction chamber for
polynucleotide analysis in one device.
In another embodiment, magnetic beads may be provided within the mesoscale
flow system, which can be moved along the flow system by an external
magnetic field, e.g., in the appliance. In one embodiment, a
polynucleotide probe may be immobilized on the magnetic beads enabling the
beads to bind to amplified polynucleotide in the reaction chamber.
Magnetic beads containing an immobilized polynucleotide probe may be,
e.g., transported through the flow system to the reaction chamber at the
end of an assay to bind to the polymerized polynucleotide product. The
bound polynucleotide may then be transported on the magnetic beads to a
detection or purification chamber in the flow system, or to a collection
port.
Some of the features and benefits of the devices are illustrated in Table
1. The devices can provide a rapid test for the detection of pathogenic
bacteria or viruses, or for the presence of certain cell types, or the
presence of a gene or a recombinant DNA sequence in a cell. The devices as
disclosed herein are all characterized by a mesoscale flow system
including a PCR chamber which is used to amplify a polynucleotide in a
sample, which may be provided with polymerase and other reagents required
for PCR. The device may be used to amplify a polynucleotide in a wide
range of applications. At the conclusion of the assay the chip typically
is discarded.
TABLE 1
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Feature Benefit
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Flexibility No limits to the number of chip
designs or applications available.
Reproducible Allows reliable, standardized, mass
production of chips.
Low Cost Allows competitive pricing with
Production existing systems. Disposable nature
for single-use processes.
Small Size No bulky instrumentation required.
Lends itself to portable units and
systems designed for use in non-
conventional lab environments.
Minimal storage and shipping costs.
Microscale Minimal sample and reagent volumes
required. Reduces reagent costs,
especially for more expensive,
specialized test procedures. Allows
simplified instrumentation schemes.
Sterility Chips can be sterilized for use in
microbiological assays and other
procedures requiring clean
environments.
Sealed System Minimizes biohazards. Ensures
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